1. Field of the Invention
The invention relates to the field of regulation of microRNA activity and expression.
2. Description of the Related Art
MicroRNAs are small RNAs that mediate gene silencing in metazoans and can regulate diverse cellular processes.
MicroRNAs (miRNAs) comprise a conserved class of small noncoding RNAs that direct targeted gene silencing through the RNA interference (RNAi) pathway in humans and other eukaryotes. Most miRNAs are encoded within long transcripts transcribed from Pol II promoters (Cai et al., 2004; Lee et al., 2002). The primary (pri-)miRNA is initially processed to an ˜65 nucleotide (nt) precursor (pre-)miRNA by the Microprocessor (Gregory et al., 2004; Han et al., 2004; Lee et al., 2003) composed of the cleaving enzyme Drosha and the RNA binding protein DGCR8. Following pri-miRNA cleavage and export from the nucleus, the pre-miRNA is processed by Dicer to a 22-25 nt miRNA duplex. One of the duplex strands termed the mature miRNA is incorporated into the RNA-induced silencing complex (RISC), which subsequently cleaves or translationally represses the target transcript depending on the degree of complementarity between the guide sequence of the miRNA and the target. miRNA-mediated gene regulation has been implicated in diverse biological processes ranging from development to angiogenesis and may be involved in the regulation of a majority of the human genome (Friedman et al., 2009).
Recently, researchers have designed synthetic RNA-based regulatory systems that integrate sensing and gene-regulatory functions, where the former are encoded in RNA aptamer sequences that recognize small molecule ligands (Suess and Weigand, 2008). Such integrated ligand-responsive RNA-based control systems offer several advantages over more traditional protein-based regulatory systems in avoiding potential immunogenicity of heterologous protein components and providing a more tunable, compact control system. In addition, as aptamers can be selected against a wide range of biomolecules (Osborne and Ellington, 1997), such integrated RNA systems provide platforms for gene expression control in response to potentially any molecular input.
Recently, integrated RNA-based control systems that mediate gene silencing through the RNAi pathway in response to small molecule ligands have been demonstrated (An et al., 2006; Beisel et al., 2008; Tuleuova et al., 2008). These systems were built from gene regulatory functions encoded by intermediate substrates in the processing pathway, small hairpin RNAs (shRNAs). Both designs linked small molecule RNA aptamers to the loop region of shRNA elements to modulate the extent of Dicer processing and subsequent gene silencing through ligand binding events based on known structural requirements for efficient Dicer processing (An et al., 2006; Beisel et al., 2008). However, the adopted mode of ligand control inherently reduced silencing (Beisel et al., 2008) and ligand regulation through Dicer processing restricts molecular sensing to cytoplasmic ligands. In addition, the expression architecture of shRNAs requires additional promoter-shRNA constructs to expand the number of ligand-responsive shRNAs, which represents a challenge for therapeutic applications. Finally, sequence restrictions and in vivo toxicity of shRNAs (Boudreau et al., 2009; Grimm et al., 2006; McBride et al., 2008) establish significant hurdles toward broader implementation of shRNA-based control systems. Other researchers have used artificial miRNAs rather than shRNAs (Bauer et al., 2009; Boudreau et al., 2009; McBride et al., 2008).
Engineered genetic systems that display ligand control of miRNA-mediated gene silencing will provide a powerful and versatile means to control transgene and endogenous gene expression.